useful to a general oxidative stress marker in the body.

Sample pretreatment procedure


8-OHdG top

Sample Pretreatment Procedure for 8-OHdG Detection.
To assay properly, please pre-treat samples desired below. In addition, please avoid repeated freeze thaw samples. In order to confirm the aptitude of assay methods on a new sample, implementing recovery test of standard 8-OHdG added into the new sample is recommended.

1. Urine Sample:
If it's clear, pretreatment is not needed. Centrifugation at 2,000~5,000g for 10 ~15 minutes is recommended for opaque samples only.
2. Serum Sample:
Blood samples must be separated to serum immediately. To separate interfering substances, filtration of serum using an ultra filter (cut off molecular weight 10,000) is necessary.
Prepare ultra-filter following manufacture's protocols.
In order to reduce deviation, diluting samples by more than twice, while paying attention to concentration range is suggested.

DNA Sample from organs and cultured cells:
It is necessary to extract samples and digest DNA before using this kit.

1. Extract DNA from cells.
Dextract DNA from the homogenates using a DNA extraction kit. Please follow the manufacture's manual.

2. Calculation of DNA Concentration and Purity.
a) DNA Solution is diluted to 20-50 micro g/mL with TE buffer.
b) Measure absorbance at wavelength 230nm, 260nm, 280nm and 320nm.
c) Calculate DNA Concentration. (1.0 of Absorbance at 260nm is equal to 50 micro g/mL DNA)
DNA concentration (micro g/mL) = (Absorbanve at 260nm) * (50 micro g/mL) * dilution rate
d) Check the purity of DNA sample.
The ratio of (Absorbance at 260nm) : (Absorbance at 280nm) is typically between 1.8 to 1.85.
The purity of isolated DNA is assessed by the ratio of (Absorbance at 260nm) : (Absorbance at 230nm), and should be between 2.2 to 2.25.

3. Enzymatic Digestion of DNA.
a) Dissolve 200 micro gram of extracted DNA in 135 micro L of water.
b) Add 15 micro L of 200mM sodium acetate and 6 units of nuclease P1 (15 micro Ll, 1 mg/mL) to the DNA Solution, and then incubated for 30 min to 1hr at 37 degree C after Argon substitution.
c) Add 1M Tris-HCl buffer (15 micro L, pH 7.4) and 2 unit of alkaline phosphatase (7 micro L, 200 units/0.7mL), and incubated for 30 min to 1hr at 37 degree C after Argon substitution.
d) To remove enzymes and other macromolecules, the hydrolysate are filtered through Millipore Ultra free C3LGC at 14000rpm for 10min.
e) Apply 50 micro L of DNA digest to the wells of ELISA kit. It is recommended that analysis procedure mentioned here shoukd be completed in one day.

4. Reagents.
a) Water: Purified Water.
b) TE buffer: 1mM EDTA in 10mM Tris-HCl(pH8.0)

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